Estradiol (17-estradiol) is a sex steroid hormone that is recognized to protect against neuronal loss caused by ischemic stroke and neurodegeneration. Protection of the aging brain will assume increasing significance with the projected graying of America's population. The prevention of neuronal loss by estradiol involves the activation of ER (estradiol receptor-) by a mechanism that is poorly understood. Data strongly suggest that activation of a sub-population of ER localized to the plasma membrane (ER was originally identified as a intracellular protein) and of the ERK (extracellular-signal regulated kinase) pathway play critical roles. Our previous publications show that (1) estradiol treatment of primary hypothalamic neurons causes the rapid trafficking of biotinylated ER into and out of the plasma membrane, a finding we corroborated in cortical neurons by showing internalization of membrane-impermeable estradiol via endosomes. (2) Estradiol treatment of primary hippocampal and cortical neurons (a) rapidly initiates GRK2 (G protein-coupled receptor kinase-2) activation, and (b) promotes the formation of ER/-arrestin-1 immuno-complexes. (3) The knockdown of - arrestin-1 using siRNA prevents estradiol-induced ERK phosphorylation. Our findings support the concept that estradiol down-regulates plasma membrane ER levels by triggering endocytosis. They also strongly suggest that the regulation of membrane ER is analogous to the regulation of rhodopsin receptor and other GPCRs. To date, the majority of laboratories studying membrane-initiated estradiol signaling have relied heavily on techniques that are subject to artifacts of fixation and tissue disruption. Deeper insight into the regulationof membrane ER should be accessible using live cells along with imaging and biophysical techniques, enabling us to examine real-time, spatially resolved events. To extend our previous work, we propose the following HYPOTHESIS: Estradiol regulates ER plasma membrane levels by a mechanism associated with receptor-mediated endocytosis. The proposed investigation builds on our previous work on estradiol action, and it introduces novel approaches to the field including real-time imaging (total internal reflection fluorescence imaging microscopy and fluorescent protein-receptor fusion constructs) and biophysical (plasma membrane capacitance measurements) techniques. The work will investigate the strong analogy between the regulation of membrane ER and regulation of rhodopsin receptors. Furthermore, the work begins to lay out a new framework for investigation of the neuroprotective effects of estradiol. The investigation will benefit from guidance by Dr. Robert Chow, an expert in single-cell imaging, biophysical measurements and membrane trafficking, and Dr. Jeannie Chen, an expert in photoreceptor physiology, especially pertaining to rhodopsin and arrestin signaling, and photoreceptor degeneration. PUBLIC HEALTH RELEVANCE: Neuronal loss, due to ischemic stroke and neurodegeneration, is attenuated by estradiol treatment. The mechanism involved in this protective action of estradiol is not completely understood but data shows the activation of ER (estrogen receptor) localized to the plasma membrane is critical. These studies seek to significantly expand our knowledge of the mechanism linking the activation of plasma membrane ER to the protection of the nervous system. !